Dissimilatory Nitrate Reduction to Ammonium (DNRA) and Denitrification Pathways Are Leveraged by Cyclic AMP Receptor Protein (CRP) Paralogues Based on Electron Donor/Acceptor Limitation in Shewanella loihica PV-4

Abstract

Under anoxic conditions, many bacteria, including Shewanella loihica strain PV-4, could use nitrate as an electron acceptor for dissimilatory nitrate reduction to ammonium (DNRA) and/or denitrification. Previous and current studies have shown that DNRA is favored under higher ambient carbon-to-nitrogen (C/N) ratios, whereas denitrification is upregulated under lower C/N ratios, which is consistent with our bioenergetics calculations. Interestingly, computational analyses indicate that the common cyclic AMP receptor protein (designated CRP1) and its paralogue CRP2 might both be involved in the regulation of two competing dissimilatory nitrate reduction pathways, DNRA and denitrification, in S. loihica PV-4 and several other denitrifying Shewanella species. To explore the regulatory mechanism underlying the dissimilatory nitrate reduction (DNR) pathways, nitrate reduction of a series of in-frame deletion mutants was analyzed under different C/N ratios. Deletion of crp1 could accelerate the reduction of nitrite to NO under both low and high C/N ratios. CRP1 is not required for denitrification and actually suppresses production of NO and N₂O gases. Deletion of either of the NO-forming nitrite reductase genes nirK or crp2 blocked production of NO gas. Furthermore, real-time PCR and electrophoretic mobility shift assays (EMSAs) demonstrated that the transcription levels of DNRA-relevant genes such as nap-β (napDABGH), nrfA, and cymA were upregulated by CRP1, while nirK transcription was dependent on CRP2. There are tradeoffs between the different physiological roles of nitrate/lactate, as nitrogen nutrient/carbon source and electron acceptor/donor and CRPs may leverage dissimilatory nitrate reduction pathways for maximizing energy yield and bacterial survival under ambient environmental conditions.IMPORTANCE Some microbes utilize different dissimilatory nitrate reduction (DNR) pathways, including DNR to ammonia (DNRA) and denitrification pathways, for anaerobic respiration in response to ambient carbon/nitrogen ratio changes. Large-scale industrial nitrogen fixation and fertilizer application raise the concern of emission of N₂O, a stable gas with potent global warming potential, as consequence of microbial respiration, thereby aggravating global warming and climate change. However, little is known about the molecular mechanism underlying the choice of two competing DNR pathways. We demonstrate that the global regulator CRP1, which is widely encoded in bacteria, is required for DNRA in S. loihica PV-4 strain, while the CRP2 paralogue is required for transcription of the nitrite reductase gene nirK for denitrification. Sufficient carbon source lead to the predominance of DNRA, while carbon source/electron donor deficiency may result in an incomplete denitrification process, raising the concern of high levels of N₂O emission from nitrate-rich and carbon source-poor waters and soils.

Keywords: Shewanella loihica; carbon-to-nitrogen ratios; cyclic AMP receptor protein; denitrification; dissimilatory nitrate reduction to ammonia.